The present invention relates to the field of molecular biology. More particularly, it relates to a novel DNA sequence having a transcriptional promoter activity, expression vectors containing this sequence, and its use for the production of recombinant proteins, and, for example, heterologous proteins. The invention also relates to recombinant cells containing this DNA sequence.
The progress made in the field of molecular biology has enabled microorganisms to be modified to make them produce heterologous proteins. In particular, numerous genetic studies have focused on the bacteria E. coli. However, the industrial application of these novel methods of production is still limited, in particular by the problems of efficiency of expression of the genes in these recombinant microorganisms. In addition, with the aim of increasing the performance of these production systems, research has been carried out in order to isolate strong promoters enabling high levels of expression of heterologous proteins to be obtained. For E. coli, the promoters of the tryptophan and lactose operons can be mentioned in particular.
More recently, with the yeast S. cerevisiae, studies have focused on promoters derived from genes implicated in glycolysis. The studies on the promoter of the gene of 3-phosphoglycerate kinase PGK (Dobson et al., Nucleic Acid Res. 10, 1982, 2625; Hitzeman et al., Nucleic Acid Research 1982, 7791), on that of the gene of glyceraldehyde-3-phosphate dehydrogenase GAPDH (Holland et al., J. Biol. Chem. 254, 1979, 9839; Musti et al., Gene 25, 1983, 133), on that of the gene of alcohol dehydrogenase 1 ADH1 (Bennentzen et al., J. Biol. Chem. 257, 1982, 3018; Denis et al., J. Biol. Chem. 25, 1983, 1165), on that of the gene of enolase 1 ENO1 (Uemura et al., Gene 45, 1986, 65), on that of the gene GAL1/GAL10 (Johnson and Davis, Mol. Cell. Biol. 4 (1984) 1440) or on that of the gene CYC1 (Guarente and Ptashne, PNAS 78 (1981) 2199) may be mentioned especially.
Recently, genetic tools have been developed so as to make use of the yeast Kluyveromyces as host cell for the production of recombinant proteins. The recognition of a two-micron type plasmid native to K. drosophilarum (plasmid PKD1xe2x80x94EP 241 435) has allowed a very efficient host/vector system for the production of recombinant proteins to be established (EP 361 991). However, the promoters used in this system have not been optimized until now. In particular, they are essentially heterologous promoters, that is to say originating from other microorganisms, such as especially S. cerevisiae. This situation can produce various disadvantages, and especially limit the activity of the promoter because of the absence of certain elements of the trans-criptional machinery (for example of trans-activators), exhibit a certain toxicity for the host cell due to an absence of regulation, or affect the stability of the vector.
Under these conditions, the lack of strong homologous promoters in Kluyveromyces constitutes a limiting factor in the industrial exploitation of this expression system.
The Applicant has now identified, cloned and sequenced a region of the genome of Kluyveromyces lactis presenting a transcriptional promoter activity (see SEQ ID No. 1 and 2). More precisely, this region corresponds to the promoter of the gene encoding the pyruvate decarboxylase of K. lactis (K1PDC1). This region, or derivatives or fragments of the latter, can be utilized in a very effective manner for the production of recombinant proteins in the yeasts of the genus Kluyveromyces. It is understood that this sequence can also be used in other host organisms.
Moreover, an advantage of the promoter region obtained lies in the absence of suppression by glucose, allowing use in conventional and industrial culture media.
One subject of the present invention therefore lies in a DNA sequence comprising all or part of the sequence SEQ ID No. 1 or of its complementary strand, or of a derivative of the latter, and possessing a transcriptional promoter activity.
In the sense of the present invention, derivative is understood as meaning any sequence obtained from the sequence SEQ ID No. 1 by modification(s) of genetic and/or chemical nature, retaining a promoter activity. Modification of genetic and/or chemical nature is understood as meaning any mutation, deletion, substitution, addition and/or modification of one or more nucleotides. Such modifications can be carried out with various aims, and especially that of preparing portable promoters, or that of preparing promoters adapted to expression in a particular type of vector or host, that of reducing the size, of increasing the activity of transcription promoter, of generating inducible promoters, of improving the level of regulation, or even of changing the nature of the regulation. Such modifications can be carried out, for example, by mutagenesis in vitro, by introduction of additional control elements or of synthetic sequences, or by deletions or substitutions of the original control elements.
When a derivative such as defined above is produced, its transcriptional promoter activity can be demonstrated in several ways, and in particular by placing under the control of the sequence studied a reporter gene whose expression is detectable. Any other technique known to the person skilled in the art can quite obviously be used to this effect.
The sequence SEQ ID No. 1 was obtained from a fusion bank between fragments of the genome of K. lactis 2359/152 and the lacZ gene of E. coli according to the protocol described in the examples. It is understood that the specialist can isolate this region by hybridization by means of a probe comprising all or part of the sequence SEQ ID No. 1 or of its complementary strand. The derivatives according to the invention can then be prepared from this sequence, as indicated in the examples.
Another object of the invention relates to a recombinant DNA comprising a sequence of DNA such as defined above.
This recombinant DNA can contain, for example, the promoter sequence SEQ ID No. 1 or a derivative of the latter in which is inserted a restriction site facilitating the use of this sequence as a xe2x80x9cportablexe2x80x9d promoter (SEQ ID No. 4).
Preferentially, this recombinant DNA in addition contains one or more structural genes. In particular, these can be genes coding for proteins of pharmaceutical or food-processing interest. By way of example, enzymes (such as, especially, superoxide dismutase, catalase, amylases, lipases, amidases, chymosin, etc.), blood derivatives (such as serum albumin, alpha- or beta-globin, factor VIII, factor IX, von Willsbrand factor, fibronectin, alpha-1 antitrypsin, etc.), insulin and its variants, lymphokines (such as interleukins, interferons, colony stimulating factors, tumor necrosis is factor (TNF) TGF-B binding vecector fragment granulocyte colony stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), and macrophage colony stimulating factor (M-CSF), etc.), growth factors (such as growth hormone, erythropoietin, fibroblast growth factor (FGF), epidermal growth factor (EFG), platelet derived growth factor (PDGF), transforming growth factor (TGF), etc.), apolipoproteins, antigenic polypeptides for the production of vaccines (hepatitis, cytomegalovirus, Epstein-Barr, herpes, etc.), or even fusions of polypeptides such as, especially, fusions comprising an active part fused to a stabilizer part (for example fusions between albumin or fragments of albumin and the receptor or a part of a virus receptor [CD4, etc.]).
Even more preferentially, the recombinant DNA also contains signals allowing the secretion of the expression product of the said structural gene(s). These signals may correspond to natural secretion signals of the protein in question, but they may be of a different origin. In particular, secretion signals derived from yeast genes can be used, such as those of the genes of the killer toxin (Stark and Boyd, EMBO J. 5 (1986) 1995) or of alpha pheromone (Kurjan and Herskowitz, Cell 30 (1982) 933; Brake et al., Yeast 4 (1988) S436).
In a particular embodiment of the invention, the recombinant DNA is part of an expression plasmid which can be of autonomous or integrative replication.
In particular, autonomous replication vectors can be obtained by using autonomous replication sequences in the chosen host. Especially, in yeast, they can be replication origins derived from plasmids (pKD1, 2xcexc, etc.), or even chromosomal sequences (ARS).
The integrative vectors can be obtained especially by using homologous sequences in certain regions of the host genome allowing, by homologous recombination, integration of the vector.
Another subject of the invention relates to recombinant cells containing a DNA sequence such as defined above.
Advantageously, the cells are chosen from amongst yeasts, and even more preferentially, amongst yeasts of the genus Kluyveromyces. It is understood, however, that the invention covers all the recombinant cells in which the promoter regions of the invention are active, whether they are eukaryotic or prokaryotic cells. Thus, among eukaryotic cells, vegetable or animal cells, yeasts or fungi can be mentioned. In particular, concerning yeasts, yeasts of the genus Saccharomyces, Pichia, Schwanniomyces or Hansenula can be mentioned. Concerning animal cells, the cells COS, CHO, C127, etc. can be mentioned. Among fungi able to be used in the present invention, Aspergillus ssp, or Trichoderma ssp, can be mentioned more particularly. As prokaryotic hosts, bacteria such as Escherichia coli can be used, or those belonging to the genera Corynebacterium, Bacillus or Streptoymces.
The transcription promoter activity of the sequences of the invention in these different hosts can be confirmed, for example, by introducing into the host cell in question a recombinant DNA comprising, under the control of the promoter sequence studied, a reporter gene whose expression can be demonstrated in the host in question.
The recombinant cells of the invention can be obtained by any method allowing a foreign DNA to he introduced into a cell. It can be especially transformation, electropotation, conjugation, fusion of protoplasts or any other technique known to the person skilled in the art. Concerning transformation, various protocols have been described in the prior art. In particular, it can be carried out by treating the whole cells in the presence of lithium acetate and of poly-ethylene glycol according to the technique described by Ito et al. (J. Bacteriol. 153 (1983) 163-168), or in the presence of ethylene glycol and dimethyl sulphoxide according to the technique of Durrens et al. (Curr. Genet. 18 (1990) 7). An alternative protocol has also been described in the Patent Application EP 361 991. Concerning electropotation, it can be carried out according to Becker and Guarentte (in: Methods in Enzymology Vol 194 (1991) 182).
Another subject of the invention relates to the use of a sequence such as defined above for the expression of recombinant genes. The DNA sequences according to the invention can in fact allow production of recombinant proteins at high levels.
Advantageously, the sequences of the invention can be used for the expression of genes encoding proteins of pharmaceutical or food-processing interest. By way of example, the proteins listed above may be mentioned.
The present invention also allows a production process for recombinant proteins to be realized, according to which a recombinant cell such as defined above is cultured and the protein produced is recovered. By way of example of protein, the proteins listed above may he mentioned.
Preferentially, the process of the invention is applicable to the production of human serum albumin, or one of its molecular variants. Molecular variant of albumin is understood as meaning the natural variants resulting from the polymorphism of the albumin, the truncated forms, or any hybrid protein based on albumin.
Other advantages of the present invention will become apparent from reading the examples which follow, which may be considered as illustrative and non-limiting.